UBIQUITIN-PROTEASOME PATHWAY IN CANCER ABSTRACT Ubiquitin-Proteasome Pathway (UPP) is a tightly regulated machinery that controls degradation and turnover of 80-90% of proteins in human cells. Its malfunction is linked to a plethora of human diseases, from neurodegenerative disorders and viral infections to cancer. Deregulation of UPP is a common theme in cancer development. The overarching goal of our research program is to develop a detailed understanding of how UPP components recognize their substrates and execute their function, which is of paramount importance for the development of new therapeutic strategies in the future. UPP components include proteasome, a cascade of ubiquitinating enzymes (E1, E2 and E3) and de- ubiquitinating enzymes (DUBs). Ubiquitinating enzymes work together to ubiquitinate their protein substrates, targeting them for proteasomal degradation. The fate of ubiquitinated proteins can be reversed by DUBs, which rescue substrates by removing ubiquitin tags. E3 ubiquitin ligases and DUBs are central to UPP because they directly interact with proteins targeted for (de)ubiquitination and determine the substrate specificity of UPP. This MIRA proposal aims to extend our successful UPP research program with a vision to structurally and functionally characterize critical ubiquitinating and de-ubiquitinating enzymes implicated in tumorigenesis in humans. Our current emphasis is on protein regulators of the p53 pathway most commonly affected in cancer. In the next five years, we will focus on the USP7 DUB enzyme and its counterpart E3 ligase Hdm2, which directly control the level of tumor suppressor p53 in a cell. Building upon extensive preliminary studies in our laboratory, which already identified a novel substrate-recognition site on USP7 and described chemical mechanisms of USP7 inhibition by several small molecule compounds, we now ask the following questions: how interactions between p53, USP7 and Hdm2 are mediated? what role USP7 conformational dynamics plays in the enzyme's function? and how can we develop new strategies to manipulate USP7 activity to stabilize p53? Here we propose (1) to take advantage of the rapidly evolving technologies in cryo-electron microscopy and determine 3D structures of macromolecular complexes formed by p53, USP7 and Hdm2, (2) to gain new insights into USP7 catalysis through a combination of structural studies and a detailed analysis of conformational dynamics of the USP7 catalytic domain using the latest advances in NMR spectroscopy, and (3) to develop new structure-based pharmaceutically relevant strategies for USP7 inhibition. The knowledge and insights gained from this research will not only unravel the molecular mechanisms by which p53 levels are regulated in a cell, but also provide new approaches to fine-tune the levels of critical protein components of the p53 and other pathways by targeting protein-specific UPP components. These results will help guide future efforts to develop novel therapeutic strategies to treat cancer and other human diseases associated with UPP malfunction. !